Field of the Invention
The present invention relates to the field of knottin mini-proteins (also known as cystine-knot peptides), and to the field of chemoselective site-specific oxime conjugation chemistry, in particular incorporating a non-natural amino acid containing an aminooxy side chain, exemplified for use in knottin mini-proteins.
Related Art
The presently exemplified peptides contain engineered (i.e. artificially created) loops having a high binding affinity for cell surface adhesion receptors (e.g. integrins) which can mediate binding to the extracellular matrix (ECM). Altered ECM interactions play an important role in tumorigenesis.1 Mediated by various receptors, these interactions are often found to enhance tumor proliferation and aggressiveness.2, 3 Integrins, a family of adhesion receptors, bind to components of ECM providing the anchorage that is necessary for cell division, migration, and invasion.4, 5 Several integrins, including αvβ3, αvβ5, and α5β1 are overexpressed in certain types of cancer and tumor vasculature, and therefore inhibitors of these integrins have generated clinical interest.6-9 
Many integrin receptors bind to an Arg-Gly-Asp (RGD) peptide motif, and the residues around it determine specificity and affinity.10 Using a RGD motif, different examples of peptides, peptidomimetics, and proteins have been developed for potential cancer therapy, but peptide scaffolds have been less explored for such application. Previously, we developed nanomolar-affinity αvβ3, αvβ5, and α5β1 integrin binding peptides by evolving a solvent exposed loop of cystine knot peptides, also known as knottins, using yeast surface display.11, 12 The presently exemplified peptides may bind all three of the above integrins, or αvβ3 only, or αvβ3/αvβ5 integrins.
Knottins have a disulfide-bonded framework and triple-stranded beta-sheet fold that often provides remarkable stability in harsh conditions, rendering them as promising candidates as pharmacophoric scaffolds for diagnostic and therapeutic applications.13, 14 Additionally, knottins are attractive for protein engineering, because the disulfide-constrained loops tolerate sequence diversity.15, 16 
Previously, low molecular weight scaffolds, including porphyrins, calixarenes, and carbohydrates have been shown to achieve orders of magnitude increase in binding strength through dimerization. However, larger scaffolds such as peptides that target integrins, have demonstrated only a several fold increase in binding strength with rarely improved therapeutic efficacy.17-25 Thus, there remains a need in the art for the development of peptides that bind integrins with significantly improved binding affinity.